Modifications to rat lens major intrinsic protein in selenite-induced cataract

Kevin L. Schey, John G. Fowler, Thomas (Tom) Shearer, Larry David

Research output: Contribution to journalArticle

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Abstract

PURPOSE. To identify modifications to rat lens major intrinsic protein (MIP) isolated from selenite-induced cataract and to determine whether m- calpain (EC 3.4.22.17) is responsible for cleavage of MIP during cataractogenesis. METHODS. Cataracts were induced in rats by a single injection of sodium selenite. Control and cataract lenses were harvested on day 16 and dissected into cortical and nuclear regions. Membranes were washed with urea buffer followed by NaOH. The protein was reduced/alkylated, delipidated, and cleaved with cyanogen bromide (CNBr). Cleavage products were fractionated by high-performance liquid chromatography (HPLC), and peptides were characterized by mass spectrometry and tandem mass spectrometry. MIP cleavage by m-calpain was carried out by incubation with purified enzyme, and peptides released from the membrane were analyzed by Edman sequencing. RESULTS. The intact C terminus, observed in the control nuclear and cataractous cortical membranes, was not observed in the cataractous nuclear membranes. Mass spectrometric analysis revealed heterogeneous cleavage of the C terminus of MIP in control and cataract nuclear regions. The major site of cleavage was between residues 238 and 239 corresponding to the major site of in vitro cleavage by m-calpain. However, sodium dodecyl sulfate- polyacrylamide gel electrophoresis and mass spectrometric analysis indicated that in vivo proteolysis during cataract formation also included sites closer to the C terminus not produced by m-calpain in vitro. Evidence for heterogeneous N-terminal cleavage was also observed at low levels with no differences between control and cataractous lenses. The major site of phosphorylation was determined to be at serine 235. CONCLUSIONS. Specific sites of MIP N- and C-terminal cleavage in selenite-induced cataractous lenses were identified. The heterogeneous cleavage pattern observed suggests that m-calpain is not the sole enzyme involved in MIP C-terminal processing in rat lens nuclei.

Original languageEnglish (US)
Pages (from-to)657-667
Number of pages11
JournalInvestigative Ophthalmology and Visual Science
Volume40
Issue number3
StatePublished - 1999

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Selenious Acid
Cataract
Lenses
Tandem Mass Spectrometry
Protein C
Proteins
Membranes
Sodium Selenite
Cyanogen Bromide
Peptides
Nuclear Envelope
Enzymes
Sodium Dodecyl Sulfate
Serine
Proteolysis
Urea
Polyacrylamide Gel Electrophoresis
Buffers
High Pressure Liquid Chromatography
Phosphorylation

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Modifications to rat lens major intrinsic protein in selenite-induced cataract. / Schey, Kevin L.; Fowler, John G.; Shearer, Thomas (Tom); David, Larry.

In: Investigative Ophthalmology and Visual Science, Vol. 40, No. 3, 1999, p. 657-667.

Research output: Contribution to journalArticle

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abstract = "PURPOSE. To identify modifications to rat lens major intrinsic protein (MIP) isolated from selenite-induced cataract and to determine whether m- calpain (EC 3.4.22.17) is responsible for cleavage of MIP during cataractogenesis. METHODS. Cataracts were induced in rats by a single injection of sodium selenite. Control and cataract lenses were harvested on day 16 and dissected into cortical and nuclear regions. Membranes were washed with urea buffer followed by NaOH. The protein was reduced/alkylated, delipidated, and cleaved with cyanogen bromide (CNBr). Cleavage products were fractionated by high-performance liquid chromatography (HPLC), and peptides were characterized by mass spectrometry and tandem mass spectrometry. MIP cleavage by m-calpain was carried out by incubation with purified enzyme, and peptides released from the membrane were analyzed by Edman sequencing. RESULTS. The intact C terminus, observed in the control nuclear and cataractous cortical membranes, was not observed in the cataractous nuclear membranes. Mass spectrometric analysis revealed heterogeneous cleavage of the C terminus of MIP in control and cataract nuclear regions. The major site of cleavage was between residues 238 and 239 corresponding to the major site of in vitro cleavage by m-calpain. However, sodium dodecyl sulfate- polyacrylamide gel electrophoresis and mass spectrometric analysis indicated that in vivo proteolysis during cataract formation also included sites closer to the C terminus not produced by m-calpain in vitro. Evidence for heterogeneous N-terminal cleavage was also observed at low levels with no differences between control and cataractous lenses. The major site of phosphorylation was determined to be at serine 235. CONCLUSIONS. Specific sites of MIP N- and C-terminal cleavage in selenite-induced cataractous lenses were identified. The heterogeneous cleavage pattern observed suggests that m-calpain is not the sole enzyme involved in MIP C-terminal processing in rat lens nuclei.",
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N2 - PURPOSE. To identify modifications to rat lens major intrinsic protein (MIP) isolated from selenite-induced cataract and to determine whether m- calpain (EC 3.4.22.17) is responsible for cleavage of MIP during cataractogenesis. METHODS. Cataracts were induced in rats by a single injection of sodium selenite. Control and cataract lenses were harvested on day 16 and dissected into cortical and nuclear regions. Membranes were washed with urea buffer followed by NaOH. The protein was reduced/alkylated, delipidated, and cleaved with cyanogen bromide (CNBr). Cleavage products were fractionated by high-performance liquid chromatography (HPLC), and peptides were characterized by mass spectrometry and tandem mass spectrometry. MIP cleavage by m-calpain was carried out by incubation with purified enzyme, and peptides released from the membrane were analyzed by Edman sequencing. RESULTS. The intact C terminus, observed in the control nuclear and cataractous cortical membranes, was not observed in the cataractous nuclear membranes. Mass spectrometric analysis revealed heterogeneous cleavage of the C terminus of MIP in control and cataract nuclear regions. The major site of cleavage was between residues 238 and 239 corresponding to the major site of in vitro cleavage by m-calpain. However, sodium dodecyl sulfate- polyacrylamide gel electrophoresis and mass spectrometric analysis indicated that in vivo proteolysis during cataract formation also included sites closer to the C terminus not produced by m-calpain in vitro. Evidence for heterogeneous N-terminal cleavage was also observed at low levels with no differences between control and cataractous lenses. The major site of phosphorylation was determined to be at serine 235. CONCLUSIONS. Specific sites of MIP N- and C-terminal cleavage in selenite-induced cataractous lenses were identified. The heterogeneous cleavage pattern observed suggests that m-calpain is not the sole enzyme involved in MIP C-terminal processing in rat lens nuclei.

AB - PURPOSE. To identify modifications to rat lens major intrinsic protein (MIP) isolated from selenite-induced cataract and to determine whether m- calpain (EC 3.4.22.17) is responsible for cleavage of MIP during cataractogenesis. METHODS. Cataracts were induced in rats by a single injection of sodium selenite. Control and cataract lenses were harvested on day 16 and dissected into cortical and nuclear regions. Membranes were washed with urea buffer followed by NaOH. The protein was reduced/alkylated, delipidated, and cleaved with cyanogen bromide (CNBr). Cleavage products were fractionated by high-performance liquid chromatography (HPLC), and peptides were characterized by mass spectrometry and tandem mass spectrometry. MIP cleavage by m-calpain was carried out by incubation with purified enzyme, and peptides released from the membrane were analyzed by Edman sequencing. RESULTS. The intact C terminus, observed in the control nuclear and cataractous cortical membranes, was not observed in the cataractous nuclear membranes. Mass spectrometric analysis revealed heterogeneous cleavage of the C terminus of MIP in control and cataract nuclear regions. The major site of cleavage was between residues 238 and 239 corresponding to the major site of in vitro cleavage by m-calpain. However, sodium dodecyl sulfate- polyacrylamide gel electrophoresis and mass spectrometric analysis indicated that in vivo proteolysis during cataract formation also included sites closer to the C terminus not produced by m-calpain in vitro. Evidence for heterogeneous N-terminal cleavage was also observed at low levels with no differences between control and cataractous lenses. The major site of phosphorylation was determined to be at serine 235. CONCLUSIONS. Specific sites of MIP N- and C-terminal cleavage in selenite-induced cataractous lenses were identified. The heterogeneous cleavage pattern observed suggests that m-calpain is not the sole enzyme involved in MIP C-terminal processing in rat lens nuclei.

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